Enzymatic synthesis of d-tagatose

ABSTRACT

The current disclosure provides a process for enzymatically converting a saccharide into tagatose. The invention also relates to a process for preparing tagatose where the process involves converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P), catalyzed by an epimerase, and converting the T6P to tagatose, catalyzed by a phosphatase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application Ser. No.62/236,226, filed on Oct. 2, 2015, which is incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates to preparation of the sugar D-tagatose. Morespecifically, the invention relates to methods of preparing D-tagatoseby enzymatically converting saccharides (e.g., polysaccharides,oligosaccharides, disaccharides, sucrose, D-glucose, and D-fructose)into D-tagatose.

BACKGROUND OF THE INVENTION

D-tagatose (tagatose hereafter) is a low-calorie, natural sweetener thathas 92% the sweetness of sucrose, but only 38% of the calories. It is anaturally occurring monosaccharide hexose that is present in only smallamounts in fruits, cacao, and dairy products. Tagatose was approved as afood additive by the Food and Drug Administration (FDA) in 2003, whichdesignated it as generally recognized as safe (GRAS). However, due totagatose's high selling prices, its use as a sweetener has been limited.Tagatose boasts a myriad of health benefits: it is non-cariogenic; it islow-calorie; it has a very low glycemic index of 3; it attenuates theglycemic index of glucose by 20%; it can lower average blood glucoselevels; it helps prevent cardiovascular disease, strokes, and othervascular diseases by raising high-density lipoprotein (HDL) cholesterol;and it is a verified prebiotic and antioxidant. Lu et al., Tagatose, aNew Antidiabetic and Obesity Control Drug, Diabetes Obes. Metab. 10(2):109-34 (2008). As such, tagatose clearly has a variety of applicationsin the pharmaceutical, biotechnological, academic, food, beverage,dietary supplement, and grocer industries.

Currently tagatose is produced predominantly through the hydrolysis oflactose by lactase or acid hydrolysis to form D-glucose and D-galactose(WO 2011150556, CN 103025894, U.S. Pat. No. 5,002,612, U.S. Pat. No.6,057,135, and U.S. Pat. No. 8,802,843). The D-galactose is thenisomerized to D-tagatose either chemically by calcium hydroxide underalkaline conditions or enzymatically by L-arabinose isomerase under pHneutral conditions. The final product is isolated by a combination offiltration and ion exchange chromatography. This process is performed inseveral tanks or bioreactors. Overall, the method suffers because of thecostly separation of other sugars (e.g., D-glucose, D-galactose, andunhydrolyzed lactose) and low product yields. Several methods viamicrobial cell fermentation are being developed, but none have beenproven to be a practical alternative due to their dependence on costlyfeedstock (e.g., galactitol and D-psicose), low product yields, andcostly separation.

There is a need to develop a cost-effective synthetic pathway forhigh-yield tagatose production where at least one step of the processinvolves an energetically favorable chemical reaction. Furthermore,there is a need for a tagatose production process where the processsteps can be conducted in one tank or bioreactor. There is also a needfor a process of tagatose production that can be conducted at arelatively low concentration of phosphate, where phosphate can berecycled, and/or the process does not require using adenosinetriphosphate (ATP) as a source of phosphate. There is also a need for atagatose production pathway that does not require the use of the costlynicotinamide adenosine dinucleotide (NAD(H)) coenzyme in any of thereaction steps.

SUMMARY OF THE INVENTION

The inventions described herein relate to processes for preparingtagatose. In various aspects, the processes involve converting fructose6-phosphate (F6P) to tagatose 6-phosphate (T6P), catalyzed by anepimerase; and converting the T6P to tagatose, catalyzed by aphosphatase. The inventions also relate to tagatose prepared by any ofthe processes described herein.

In some aspects of the invention, a process for preparing tagatose alsoinvolves the step of converting glucose 6-phosphate (G6P) to the F6P,where the step is catalyzed by phosphoglucose isomerase (PGI). In otheraspects, a process for tagatose synthesis also includes the step ofconverting glucose 1-phosphate (G1P) to the G6P, and this conversionstep is catalyzed by phosphoglucomutase (PGM).

In various aspects, a process for preparing tagatose can involveconverting a saccharide to the G1P, catalyzed by at least one enzyme;converting G1P to G6P, catalyzed by phosphoglucomutase (PGM); convertingG6P to F6P, catalyzed by phosphoglucose isomerase (PGI); converting F6Pto tagatose 6-phosphate (T6P), catalyzed by an epimerase; and convertingthe T6P produced to tagatose, catalyzed by a phosphatase.

The saccharides used in any of the processes can be selected from thegroup consisting of a starch or its derivative, cellulose or itsderivative, and sucrose. The starch or its derivative can be amylose,amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, orglucose. In some aspects of the invention, a process for preparingtagatose involves converting starch to a starch derivative by enzymatichydrolysis or by acid hydrolysis of starch. In other aspects, a starchderivative can be is prepared by enzymatic hydrolysis of starchcatalyzed by isoamylase, pullulanase, alpha-amylase, or a combination oftwo or more of these enzymes. A process for preparing tagatose, incertain aspects, can also involve adding 4-glucan transferase (4GT).

In various aspects, a process for preparing tagatose can involveconverting fructose to the F6P, catalyzed by at least one enzyme;converting F6P to tagatose 6-phosphate (T6P) catalyzed by an epimerase;and converting the T6P produced to tagatose, catalyzed by a phosphatase.In other embodiments, a tagatose production process involves convertingsucrose to the fructose, catalyzed by at least one enzyme; convertingfructose to the F6P, catalyzed by at least one enzyme; converting F6P totagatose 6-phosphate (T6P) catalyzed by an epimerase; and converting theT6P produced to tagatose, catalyzed by a phosphatase.

In other aspects of the invention, G6P to be used in a process forpreparing tagatose can be generated by converting glucose to the G6P,catalyzed by at least one enzyme. Glucose can in turn be produced byconverting sucrose to glucose, catalyzed by at least one enzyme.

In some aspects of the invention, epimerase used to convert F6P to T6Pis fructose 6-phosphate epimerase. The fructose 6-phosphate epimerasecan be encoded by a polynucleotide comprising a nucleotide sequencehaving at least 60%, at least 70%, at least 80%, at least 85%, at least90%, at least 95%, at least 97%, at least 99%, or 100% sequence identitywith SEQ ID NOS.: 1, 3, 5, 7, 9, or 10. In various aspects, the fructose6-phosphate epimerase comprises an amino acid sequence having at least60%, at least 70%, at least 80%, at least 85%, at least 90%, at least95%, at least 97%, at least 99%, or 100% sequence identity with SEQ IDNOS.: 2, 4, 6, 8, or 11.

In various aspects of the invention, the phosphatase used to convert T6Pto tagatose is tagatose 6-phosphate phosphatase. The tagatose6-phosphate phosphatase can be encoded by a polynucleotide comprising anucleotide sequence having at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99%, or100% sequence identity with SEQ ID NO.: 12, 14, or 16. In some aspectsof the invention, the tagatose 6-phosphate phosphatase comprises anamino acid sequence having at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99%, or100% sequence identity with SEQ ID NO.: 13, 15, or 17.

In various aspects, a process of the invention are be conducted at atemperature ranging from about 40° C. to about 70° C., at a pH rangingfrom about 5.0 to about 8.0, and/or for about 8 hours to about 48 hours.In some aspects, the steps of a process for preparing tagatose areconducted in one bioreactor. In other aspects, the steps are conductedin a plurality of bioreactors arranged in series.

In other aspects of the invention, the steps of a process for preparingtagatose are conducted ATP-free, NAD(H)-free, at a phosphateconcentration from about 0 mM to about 150 mM, the phosphate isrecycled, and/or at least one step of the process involves anenergetically favorable chemical reaction.

BRIEF DESCRIPTION OF THE FIGURES

These drawings illustrate certain aspects of some of the embodiments ofthe invention, and should not be used to limit or define the invention.

FIG. 1 is a schematic diagram illustrating an enzymatic pathwayconverting fructose 6-phosphate to tagatose 6-phosphate and then toD-tagatose (tagatose).

FIG. 2 is a schematic diagram illustrating an enzymatic pathwayconverting starch or its derived products to tagatose. The followingabbreviations are used: αGP, alpha-glucan phosphorylase or starchphosphorylase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase;F6PE, fructose 6-phosphate epimerase; T6PP, tagatose 6-phosphatephosphatase; IA, isoamylase; PA, pullulanase; MP, maltose phosphorylase;PPGK, polyphosphate glucokinase.

FIG. 3 shows an enzymatic pathway converting cellulose or its derivedproducts to tagatose. CDP, cellodextrin phosphorylase; CBP, cellobiosephosphorylase; PPGK, polyphosphate glucokinase; PGM, phosphoglucomutase;PGI, phosphoglucoisomerase; F6PE, fructose 6-phosphate epimerase; T6PP,tagatose 6-phosphate phosphatase.

FIG. 4 is a schematic diagram illustrating an enzymatic pathwayconverting fructose to tagatose. PPFK, polyphosphate fructokinase; F6PE,fructose 6-phosphate epimerase; T6PP, tagatose 6-phosphate phosphatase.

FIG. 5 is a schematic diagram illustrating an enzymatic pathwayconverting glucose to tagatose. PPGK, polyphosphate glucokinase; PGI,phosphoglucoisomerase; F6PE, fructose 6-phosphate epimerase; T6PP,tagatose 6-phosphate phosphatase.

FIG. 6 shows an enzymatic pathway converting sucrose or its derivedproducts to tagatose. SP, sucrose phosphorylase; PPFK, polyphosphatefructokinase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase; F6PE,fructose 6-phosphate epimerase; T6PP, tagatose 6-phosphate phosphatase.

FIG. 7 shows the Reaction Gibbs Energy between intermediates based onformation Gibbs energy for the conversion of glucose 1-phosphate totagatose.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides enzymatic pathways, or processes, forsynthesizing tagatose with a high product yield, while greatlydecreasing the product separation costs and tagatose production costs.

The invention relates to a process for preparing tagatose where theprocess involves converting fructose 6-phosphate (F6P) to tagatose6-phosphate (T6P) catalyzed by an epimerase and converting the T6Pproduced to tagatose catalyzed by a phosphatase (e.g., tagatose6-phosphate phosphatase, T6PP). This process is generally shown inFIG. 1. In certain embodiments, the epimerase that catalyzes theconversion of F6P to T6P is fructose 6-phosphate epimerase (F6PE).

Epimerases that convert F6P to T6P may be used in a process of theinvention. In some aspects of the invention, epimerases suitable for usein the processes to convert F6P to T6P comprise an amino acid sequencethat has a degree of identity to the amino acid sequence of SEQ ID NOS.:2, 4, 6, 8, or 11 (shown below), of at least 60%, preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, 97%, 98%, 99%, or 100%. The suitable epimerasesare encoded by a polynucleotide comprising a nucleotide sequence thathas a degree of identity to the nucleotide sequence of SEQ ID NOS.: 1,3, 5, 7, 9, or 10 (shown below), of at least 60%, preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, 97%, 98%, 99%, or 100%.

The invention also relates to epimerases that comprise an amino acidsequence that has a degree of identity to the amino acid sequence of SEQID NOS.: 2, 4, 6, 8, or 11, of at least 60%, preferably at least 65%,more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, 97%, 98%, 99%, or 100%. In other aspects, theinvention relates to epimerases that are encoded by a polynucleotidecomprising a nucleotide sequence that has a degree of identity to thenucleotide sequence of SEQ ID NOS.: 1, 3, 5, 7, 9, or 10, of at least60%, preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 96%, 97%, 98%, 99%, or100%.

Phosphatases that convert T6P to tagatose (D-tagatose) may be used in aprocess of the invention. In some aspects of the invention, phosphatasesthat can be used in to convert T6P to tagatose (D-tagatose) comprise anamino acid sequence that has a degree of identity to the amino acidsequence of SEQ ID NOS.: 12, 14, or 16 (shown below), of at least 60%,preferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, 97%, 98%, 99%, or 100%. Thetagatose phosphatases are encoded by a polynucleotide comprising anucleotide sequence that has a degree of identity to the nucleotidesequence of SEQ ID NOS.: 13, 15, or 17 (shown below), of at least 60%,preferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, 97%, 98%, 99%, or 100%.

The invention also relates to phosphatases that convert T6P to tagatose(D-tagatose) and comprise an amino acid sequence that has a degree ofidentity to the amino acid sequence of SEQ ID NOS.: 12, 14, or 16, of atleast 60%, preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 96%, 97%, 98%, 99%, or100%. In various aspects, the invention relates to phosphatases thatconvert T6P to tagatose (D-tagatose) and are encoded by a polynucleotidecomprising a nucleotide sequence that has a degree of identity to thenucleotide sequence of SEQ ID NOS.: 13, 15, or 17, of at least 60%,preferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, 97%, 98%, 99%, or 100%.

In some embodiments, a process for preparing tagatose according to theinvention also includes the step of enzymatically converting glucose6-phosphate (G6P) to the F6P, and this step is catalyzed byphosphoglucose isomerase (PGI). In other embodiments, the process forpreparing tagatose additionally includes the step of converting glucose1-phosphate (G1P) to the G6P, where the step is catalyzed byphosphoglucomutase (PGM). In yet further embodiments, tagatoseproduction process also includes the step of converting a saccharide tothe G1P that is catalyzed at least one enzyme.

Therefore, a process for preparing tagatose according to the inventioncan, for example, include the following steps: (i) converting asaccharide to glucose 1-phosphate (G1P) using one or more enzymes; (ii)converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii)converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9);(iv) converting F6P to T6P via fructose 6-phosphate epimerase (F6PE),and (v) converting T6P to tagatose via tagatose 6-phosphate phosphatase(T6PP). An example of the process where the saccharide is starch isshown in FIG. 2.

Typically, the ratios of enzyme units used in the disclosed process are1:1:1:1:1 (αGP:PGM:PGI:F6PE:T6PP). To optimize product yields, theseratios can be adjusted in any number of combinations. For example, aratio of 3:1:1:1:1 can be used to maximize the concentration ofphosphorylated intermediates, which will result in increased activity ofthe downstream reactions. Conversely, a ratio of 1:1:1:1:3 can be usedto maintain a robust supply of phosphate for αGP, which will result inmore efficient phosphorolytic cleavage of alpha-1,4-glycosidic bonds. Aratio of enzymes, for example, 3:1:1:1:3 can be used to further increasethe reaction rate. Therefore, the enzyme ratios, including otheroptional enzymes discussed below, can be varied to increase theefficiency of tagatose production. For example, a particular enzyme maybe present in an amount about 2×, 3×, 4×, 5×, etc. relative to theamount of other enzymes.

One of the important advantages of the processes is that the processsteps can be conducted in one bioreactor or reaction vessel.Alternatively, the steps can also be conducted in a plurality ofbioreactors, or reaction vessels, that are arranged in series.

Phosphate ions produced by T6PP dephosphorylation of T6P can then berecycled in the process step of converting a saccharide to G1P,particularly when all process steps are conducted in a single bioreactoror reaction vessel. The ability to recycle phosphate in the disclosedprocesses allows for non-stoichiometric amounts of phosphate to be used,which keeps reaction phosphate concentrations low. This affects theoverall pathway and the overall rate of the processes, but does notlimit the activity of the individual enzymes and allows for overallefficiency of the tagatose making processes.

For example, reaction phosphate concentrations can range from about 0 mMto about 300 mM, from about 0 mM to about 150 mM, from about 1 mM toabout 50 mM, preferably from about 5 mM to about 50 mM, or morepreferably from about 10 mM to about 50 mM. For instance, the reactionphosphate concentration can be about 0.1 mM, about 0.5 mM, about 1 mM,about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM,or about 55 mM.

Therefore, low phosphate concertation results in decreased productioncosts due to low total phosphate and thus lowered cost of phosphateremoval. It also prevents inhibition of T6PP by high concentrations offree phosphate and decreases the potential for phosphate pollution.

Furthermore, the processes disclosed herein can be conducted withoutadded ATP as a source of phosphate, i.e., ATP-free. The processes canalso be conducted without having to add NAD(H), i.e., NAD(H)-free. Otheradvantages also include the fact that at least one step of the disclosedprocesses for making tagatose involves an energetically favorablechemical reaction (FIG. 7).

Examples of the enzymes used to convert a saccharide to G1P includealpha-glucan phosphorylase (αGP, EC 2.4.1.1), maltose phosphorylase (MP,EC 2.4.1.8), cellodextrin phosphorylase (CDP, EC 2.4.1.49), cellobiosephosphorylase (CBP, EC 2.4.1.20), cellulose phosphorylase, sucrosephosphorylase (SP, EC 2.4.1.7), and a combination thereof. The choice ofthe enzyme or enzyme combination depends on the saccharide used in theprocess.

The saccharides used for generating G1P can be polysaccharides,oligosaccharides, and/or disaccharides. For example, the saccharide canbe starch, one or more derivatives of starch, cellulose, one or morederivatives of cellulose, sucrose, one or more derivatives of sucrose,or a combination thereof.

Starch is the most widely used energy storage compound in nature and ismostly stored in plant seeds. Natural starch contains linear amylose andbranched amylopectin. Examples of starch derivatives include amylose,amylopectin, soluble starch, amylodextrin, maltodextrin, maltose,fructose, and glucose. Examples of cellulose derivatives includepretreated biomass, regenerated amorphous cellulose, cellodextrin,cellobiose, fructose, and glucose. Sucrose derivatives include fructoseand glucose.

The derivatives of starch can be prepared by enzymatic hydrolysis ofstarch or by acid hydrolysis of starch. Specifically, the enzymatichydrolysis of starch can be catalyzed or enhanced by isoamylase (IA, EC.3.2.1.68), which hydrolyzes α-1,6-glucosidic bonds; pullulanase (PA, EC.3.2.1.41), which hydrolyzes α-1,6-glucosidic bonds;4-α-glucanotransferase (4GT, EC. 2.4.1.25), which catalyzes thetransglycosylation of short maltooligosaccharides, yielding longermaltooligosaccharides; or alpha-amylase (EC 3.2.1.1), which cleavesα-1,4-glucosidic bonds.

Furthermore, derivatives of cellulose can be prepared by enzymatichydrolysis of cellulose catalyzed by cellulase mixtures, by acids, or bypretreatment of biomass.

In certain embodiments, the enzymes used to convert a saccharide to G1Pcontain αGP. In this step, when the saccharides include starch, the G1Pis generated from starch by αGP; when the saccharides contain solublestarch, amylodextrin, or maltodextrin, the G1P is produced from solublestarch, amylodextrin, or maltodextrin by αGP.

When the saccharides include maltose and the enzymes contain maltosephosphorylase, the G1P is generated from maltose by maltosephosphorylase. If the saccharides include sucrose, and enzymes containsucrose phosphorylase, the G1P is generated from sucrose by sucrosephosphorylase.

In yet another embodiment, when the saccharides include cellobiose, andthe enzymes contain cellobiose phosphorylase, the G1P is generated fromcellobiose by cellobiose phosphorylase.

In an additional embodiment, when the saccharides contain cellodextrinsand the enzymes include cellodextrin phosphorylase, the G1P is generatedfrom cellodextrins by cellodextrin phosphorylase.

In an alternative embodiment of converting a saccharide to G1P, when thesaccharides include cellulose, and enzymes contain cellulosephosphorylase, the G1P is generated from cellulose by cellulosephosphorylase.

According to the invention, tagatose can also be produced from fructose.An example of the process is shown in FIG. 4. For example, the processinvolves generating F6P from fructose and polyphosphate catalyzed bypolyphosphate fructokinase (PPFK); converting F6P to T6P catalyzed byF6PE; and converting T6P to tagatose catalyzed by T6PP. The fructose canbe produced, for example, by an enzymatic conversion of sucrose.

In other embodiments, tagatose can be produced from sucrose. An exampleof such process is shown in FIG. 6. The process provides an in vitrosynthetic pathway that includes the following enzymatic steps:generating G1P from sucrose and free phosphate catalyzed by sucrosephosphorylase (SP); converting G1P to G6P catalyzed by PGM; convertingG6P to F6P catalyzed by PGI; converting F6P to T6P catalyzed by F6PE;and converting T6P to tagatose catalyzed by T6PP.

The phosphate ions generated when T6P is converted to tagatose can thenbe recycled in the step of converting sucrose to G1P. Additionally, asshown in FIG. 6, PPFK and polyphosphate can be used to increase tagatoseyields by producing F6P from fructose generated by the phosphorolyticcleavage of sucrose by SP.

In some embodiments, a process for preparing tagatose includes thefollowing steps: generating glucose from polysaccharides andoligosaccharides by enzymatic hydrolysis or acid hydrolysis, convertingglucose to G6P catalyzed by at least one enzyme, generating fructosefrom polysaccharides and oligosaccharides by enzymatic hydrolysis oracid hydrolysis, and converting fructose to G6P catalyzed by at leastone enzyme. Examples of the polysaccharides and oligosaccharides areenumerated above.

In other embodiments, G6P is produced from glucose and sodiumpolyphosphate by polyphosphate glucokinase.

The present disclosure provides processes for converting saccharides,such as polysaccharides and oligosaccharides in starch, cellulose,sucrose and their derived products, to tagatose. In certain embodiments,artificial (non-natural) ATP-free enzymatic pathways are provided toconvert starch, cellulose, sucrose, and their derived products totagatose using cell-free enzyme cocktails.

As shown above, several enzymes can be used to hydrolyze starch toincrease the G1P yield. Such enzymes include isoamylase, pullulanase,and alpha-amylase. Corn starch contains many branches that impede αGPaction. Isoamylase can be used to de-branch starch, yielding linearamylodextrin. Isoamylase-pretreated starch can result in a higher F6Pconcentration in the final product. Isoamylase and pullulanase cleavealpha-1,6-glycosidic bonds, which allows for more complete degradationof starch by alpha-glucan phosphorylase. Alpha-amylase cleavesalpha-1,4-glycosidic bonds, therefore alpha-amylase is used to degradestarch into fragments for quicker conversion to tagatose.

As shown in FIG. 2, maltose phosphorylase (MP) can be used to increasetagatose yields by phosphorolytically cleaving the degradation productmaltose into G1P and glucose. Alternatively, 4-glucan transferase (4GT)can be used to increase tagatose yields by recycling the degradationproducts glucose, maltose, and maltotriose into longermaltooligosaccharides; which can be phosphorolytically cleaved by αGP toyield G1P.

Additionally, cellulose is the most abundant bio resource and is theprimary component of plant cell walls. Non-food lignocellulosic biomasscontains cellulose, hemicellulose, and lignin as well as other minorcomponents. Pure cellulose, including Avicel (microcrystallinecellulose), regenerated amorphous cellulose, bacterial cellulose, filterpaper, and so on, can be prepared via a series of treatments. Thepartially hydrolyzed cellulosic substrates include water-insolublecellodextrins whose degree of polymerization is more than 7,water-soluble cellodextrins with degree of polymerization of 3-6,cellobiose, glucose, and fructose.

In certain embodiments, cellulose and its derived products can beconverted to tagatose through a series of steps. An example of suchprocess is a shown in FIG. 3. The process provides an in vitro syntheticpathway that involves the following steps: generating G1P fromcellodextrin and cellobiose and free phosphate catalyzed by cellodextrinphosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively;converting G1P to G6P catalyzed by PGM; converting G6P to F6P catalyzedby PGI; converting F6P to T6P catalyzed by F6PE; and converting T6P totagatose catalyzed by T6PP. In this process, the phosphate ions can berecycled by the step of converting cellodextrin and cellobiose to G1P.

Several enzymes may be used to hydrolyze solid cellulose towater-soluble cellodextrins and cellobiose. Such enzymes includeendoglucanase and cellobiohydrolase, but not including beta-glucosidase(cellobiase).

Prior to cellulose hydrolysis and G1P generation, cellulose and biomasscan be pretreated to increase their reactivity and decrease the degreeof polymerization of cellulose chains.

Cellulose and biomass pretreatment methods include dilute acidpretreatment, cellulose solvent-based lignocellulose fractionation,ammonia fiber expansion, ammonia aqueous soaking, ionic liquidtreatment, and partially hydrolyzed by using concentrated acids,including hydrochloric acid, sulfuric acid, phosphoric acid and theircombinations.

In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK)can be added to the process, thus increasing yields of tagatose byphosphorylating the degradation product glucose to G6P, as shown in FIG.3.

In other embodiments, tagatose can be generated from glucose. An exampleof such process is shown in FIG. 5. The process involves the steps ofgenerating G6P from glucose and polyphosphate catalyzed by polyphosphateglucokinase (PPGK); converting G6P to F6P catalyzed by PGI; convertingF6P to T6P catalyzed by F6PE; and converting T6P to tagatose catalyzedby T6PP.

Any suitable biological buffer known in the art can be used in a processof the invention, such as HEPES, PBS, BIS-TRIS, MOPS, DIPSO, Trizma,etc. The reaction buffer for all embodiments can have a pH ranging from5.0-8.0. More preferably, the reaction buffer pH can range from about6.0 to about 7.3. For example, the reaction buffer pH can be 6.0, 6.2,6.4, 6.6, 6.8, 7.0, 7.2, or 7.3.

The reaction buffer can also contain key metal cations. Examples of themetal ions include Mg²⁺ and Zn²⁺.

The reaction temperature at which the process steps are conducted canrange from 37-85° C. More preferably, the steps can be conducted at atemperature ranging from about 40° C. to about 70° C. The temperaturecan be, for example, about 40° C., about 45° C., about 50° C., about 55°C., or about 60° C. Preferably, the reaction temperature is about 50° C.

The reaction time of the disclosed processes can be adjusted asnecessary, and can range from about 8 hours to about 48 hours. Forexample, the reaction time can be about 16 hours, about 18 hours, about20 hours, about 22 hours, about 24 hours, about 26 hours, about 28hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours,about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46hours, or about 48 hours. More preferably, the reaction time is about 24hours.

The processes according to the invention can achieve high yields due tothe very favorable equilibrium constant for the overall reaction. Forexample, FIG. 7 shows the Reaction Gibbs Energy between intermediatesbased on formation Gibbs energy for the conversion of glucose1-phosphate to tagatose. Reaction Gibbs Energies were generated usinghttp://equilibrator.weizmann.ac.il/. Theoretically, up to 99% yields canbe achieved if the starting material is completely converted to anintermediate.

Processes of the invention use low-cost starting materials and reduceproduction costs by decreasing costs associated with the feedstock andproduct separation. Starch, cellulose, sucrose and their derivatives areless expensive feedstocks than, for example, lactose. When tagatose isproduced from lactose, glucose and galactose and tagatose are separatedvia chromatography, which leads to higher production costs.

Also, the step of converting T6P to tagatose according to the inventionis an irreversible phosphatase reaction, regardless of the feedstock.Therefore, tagatose is produced with a very high yield while effectivelyminimizing the subsequent product separation costs.

In contrast to cell-based manufacturing methods, the invention involvesa cell-free preparation of tagatose, has relatively high reaction ratesdue to the elimination of the cell membrane, which often slows down thetransport of substrate/product into and out of the cell.

It also has a final product free of nutrient-rich fermentationmedia/cellular metabolites.

EXAMPLES Materials and Methods

Chemicals

All chemicals, including corn starch, soluble starch, maltodextrins,maltose, glucose, filter paper were reagent grade or higher andpurchased from Sigma-Aldrich (St. Louis, Mo., USA) or Fisher Scientific(Pittsburgh, Pa., USA), unless otherwise noted. Restriction enzymes, T4ligase, and Phusion DNA polymerase were purchased from New EnglandBiolabs (Ipswich, Mass., USA). Oligonucleotides were synthesized eitherby Integrated DNA Technologies (Coralville, Iowa, USA) or Eurofins MWGOperon (Huntsville, Ala., USA). Regenerated amorphous cellulose used inenzyme purification was prepared from Avicel PH105 (FMC BioPolymer,Philadelphia, Pa., USA) through its dissolution and regeneration, asdescribed in: Ye et al., Fusion of a family 9 cellulose-binding moduleimproves catalytic potential of Clostridium thermocellum cellodextrinphosphorylase on insoluble cellulose. Appl. Microbiol. Biotechnol. 2011;92:551-560. Escherichia coli Sig10 (Sigma-Aldrich, St. Louis, Mo., USA)was used as a host cell for DNA manipulation and E. coli BL21 (DE3)(Sigma-Aldrich, St. Louis, Mo., USA) was used as a host cell forrecombinant protein expression. ZYM-5052 media including either 100 mgL⁻¹ ampicillin or 50 mg L⁻¹ kanamycin was used for E. coli cell growthand recombinant protein expression. Cellulase from Trichoderma reesei(Catalog number: C2730) and pullulanase (Catalog number: P1067) werepurchased from Sigma-Aldrich (St. Louis, Mo., USA) and produced byNovozymes (Franklinton, N.C., USA). Maltose phosphorylase (Catalognumber: M8284) was purchased from Sigma-Aldrich.

Production and Purification of Recombinant Enzymes

The E. coli BL21 (DE3) strain harboring a protein expression plasmid wasincubated in a 1-L Erlenmeyer flask with 100 mL of ZYM-5052 mediacontaining either 100 mg L⁻¹ ampicillin or 50 mg L⁻¹ kanamycin. Cellswere grown at 37° C. with rotary shaking at 220 rpm for 16-24 hours. Thecells were harvested by centrifugation at 12° C. and washed once witheither 20 mM HEPES (pH 7.5) containing 50 mM NaCl and 5 mM MgCl₂ (heatprecipitation and cellulose-binding module) or 20 mM HEPES (pH 7.5)containing 300 mM NaCl and 5 mM imidazole (Ni purification). The cellpellets were re-suspended in the same buffer and lysed byultra-sonication (Fisher Scientific Sonic Dismembrator Model 500; 5 spulse on and 10 s off, total 21 min at 50% amplitude). Aftercentrifugation, the target proteins in the supernatants were purified.

Three approaches were used to purify the various recombinant proteins.His-tagged proteins were purified by the Profinity IMAC Ni-Charged Resin(Bio-Rad, Hercules, Calif., USA). Fusion proteins containing acellulose-binding module (CBM) and self-cleavage intein were purifiedthrough high-affinity adsorption on a large surface-area regeneratedamorphous cellulose. Heat precipitation at 70-95° C. for 5-30 min wasused to purify hyperthermostable enzymes. The purity of the recombinantproteins was examined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE).

Enzymes Used and their Activity Assays

Alpha-glucan phosphorylase (αGP) from Thermotoga maritima (Uniprot IDG4FEH8) was used. Activity was assayed in 50 mM sodium phosphate buffer(pH 7.2) containing 1 mM MgCl₂, 5 mM DTT, and 30 mM maltodextrin at 50°C. The reaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO) (Vivaproducts, Inc., Littleton, Mass., USA).Glucose 1-phosphate (G1P) was measured using a glucose hexokinase/G6PDHassay kit (Sigma Aldrich, Catalog No. GAHK20-1KT) supplemented with 25U/mL phosphoglucomutase. A unit (U) is described as mol/min.

Phosphoglucomutase (PGM) from Thermococcus kodakaraensis (Uniprot IDQ68BJ6) was used. Activity was measured in 50 mM HEPES buffer (pH 7.2)containing 5 mM MgCl₂ and 5 mM G1P at 50° C. The reaction was stoppedvia filtration of enzyme with a Vivaspin 2 concentrator (10,000 MWCO).The product glucose 6-phosphate (G6P) was determined using ahexokinase/G6PDH assay kit (Sigma Aldrich, Catalog No. GAHK20-1KT).

Two different sources of phosphoglucoisomerase (PGI) were used fromClostridium thermocellum (Uniprot ID A3DBX9) and Thermus thermophilus(Uniprot ID Q5SLL6). Activity was measured in 50 mM HEPES buffer (pH7.2) containing 5 mM MgCl₂ and 10 mM G6P at 50° C. The reaction wasstopped via filtration of enzyme with a Vivaspin 2 concentrator (10,000MWCO). The product, fructose 6-phosphate (F6P), was determined using afructose 6-phosphate kinase (F6PK)/pyruvate dehydrogenase (PK)/lactatedehydrogenase (LD) coupled enzyme assay where a decrease in absorbanceat 340 nm indicates production of F6P. This 200 μL reaction contained 50mM HEPES (pH 7.2), 5 mM MgCl₂, 10 mM G6P, 1.5 mM ATP, 1.5 mM phosphoenolpyruvate, 200 μM NADH, 0.1 U PGI, 5 U PK, and 5 U LD.

Fructose 6-phosphate epimerase (F6PE) from Dictyoglomus thermophilum(Uniprot ID B5YBD7) was used. Activity was measured in 50 mM HEPESbuffer (pH 7.2) containing 5 mM MgCl₂, and 10 mM F6P at 50° C. Thereaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). The product, tagatose 6-phosphate (T6P), wasdetermined using tagatose 6-phosphate phosphatase and detecting freephosphate release. To detect free phosphate release, 500 μL of asolution containing 0.1 M zinc acetate and 2 mM ammonium molybdate (pH5) was added to 50 μL of reaction. This was mixed and followed by 125 μLof 5% ascorbic acid (pH 5). This solution was mixed then incubated at30° C. for 20 min. The absorbance at 850 nm was read to determine freephosphate release.

Thermophilic F6PE from Anaerolinea thermophila UNI-1 (Uniprot ID:E8N0N6)

Nucleotide Sequence (SEQ ID NO.: 1)ATGTTCGGCTCGCCTGCTCCCCTGCTGGATATGGTCACCGCGCAGAAACAGGGCATGGCGCGGGGTATCCCATCCATTTGTTCGGCACATCCGGTGGTGCTGAGTGCCGCCTGCCATCTTGCCCGCCGGAGCGGCGCGCCCCTGCTCATCGAAACCACCTGCAATCAGGTCAACCACCAAGGTGGGTACAGCGGCATGACCCCCGCCGATTTTGTCCGCTTTCTGCGCGAAATTCTGGAACGGGAAGGTATTCCCCCGCAACAGGTCATCCTGGGCGGGGATCACCTGGGTCCTTACCCCTGGCGGAAAGAGCCTGCCGAAACCGCCATAGCACAAGCGCTGGAAATGGTGCGGGCATACGTGCAGGCAGGCTACACCAAAATTCATCTGGACGCTTCCATGCCCTGCGCCGATGACGACCCCGAGCGTCCCCTGCCGCTGGAGCGCATAGCCCGACGGGCGGCGCAGTTGTGCGCCGCCGCCGAAGCCGCCGCGGGAGCGGTTCAGCCGGTGTACGTAATTGGCAGTGAGGTGCCCCCGCCCGGCGGCGCGCAGGGTCAGGAGGCAAGACTTCACGTCACCACTCCGCAGGAAGCCCAAGCCGCGCTGGATGCCTTTCGGGAAGCCTTTCTGCAGGCAGGCTTGACTCCCGTTTGGGAGCGGGTCATTGCGCTGGTAGTCCAGCCGGGGGTGGAGTTTGGCGTGGACAGCATTCACGCCTATCAGCGCGAAGCCGCCCGCCCGCTGAAGACCTTCATCGAGGGCGTGCCCGGCATGGTGTATGAAGCCCACTCGACCGATTACCAGACCCGTGCCTCCCTGCGTGCGCTGGTGGAAGACCACTTTTCCATTCTCAAGGTTGGTCCGGCACTAACCTTTGCCTACCGCGAAGCCGTGTTCGCCCTGGAACACATCGAACGGGAAATATTGGGCAGGCAGGATATGCCTCTCTCCCGCCTGAGTGAAGTCCTCGACGAGGTGATGCTGAACGATCCACGCCACTGGCAGGGATACTTTGCCGGCGCTCCCGCCGAACAGGCGCTGGCGCGCCGCTACAGTTTCAGCGACCGCATTCGCTATTACTGGCACCATCCCGCCGCGCAGGAAGCCGTGCGGAGACTGCTCGCCAACCTGATCGAAACCCCGCCGCCGCTGAGTTTGCTCAGCCAGTACCTGCCGCGCGAGTATGAGATGGTGCGCGCGGGGGAAATCTCCAGCCACCCGCAGGACCTGATTCGGGCACATATCCAGCACACGCTGGAAGATTACGCTGCGGCGTGCGGGTAA Amino acid sequence(SEQ ID NO.: 2) MFGSPAPLLDMVTAQKQGMARGIPSICSAHPVVLSAACHLARRSGAPLLIETTCNQVNHQGGYSGMTPADFVRFLREILEREGIPPQQVILGGDHLGPYPWRKEPAETAIAQALEMVRAYVQAGYTKIHLDASMPCADDDPERPLPLERIARRAAQLCAAAEAAAGAVQPVYVIGSEVPPPGGAQGQEARLHVTTPQEAQAALDAFREAFLQAGLTPVWERVIALVVQPGVEFGVDSIHAYQREAARPLKTFIEGVPGMVYEAHSTDYQTRASLRALVEDHFSILKVGPALTFAYREAVFALEHIEREILGRQDMPLSRLSEVLDEVMLNDPRHWQGYFAGAPAEQALARRYSFSDRIRYYWHHPAAQEAVRRLLANLIETPPPLSLLSQYLPREYEMVRAGEISSHPQDLIRA HIQHTLEDYAAACG

Thermophilic F6PE from Caldicellulosiruptor kronotskyensis (Uniprot ID:E4SEH3)

Nucleotide sequence (SEQ ID NO.: 3)ATGAGTCCTCAAAATCCATTGATTGGTTTATTTAAGAATAGAGAAAAAGAGTTTAAGGGTATTATTTCAGTTTGTTCTTCAAATGAAATAGTCTTAGAAGCAGTTTTAAAAAGAATGAAAGATACAAACCTACCAATTATTATTGAAGCCACAGCGAACCAGGTAAATCAATTTGGCGGGTATTCTGGGTTGACACCGTCTCAGTTCAAAGAACGAGTTATAAAAATTGCTCAAAAAGTTGATTTTCCACTTGAGAGAATAATTCTTGGTGGGGACCATCTTGGACCATTTGTGTGGCGTGACCAGGAACCAGAAATTGCTATGGAGTATGCTAAGCAAATGATAAAAGAATACATAAAAGCAGGTTTTACCAAAATTCACATCGACACGAGTATGCCTTTAAAAGGGGAGAACAGCATAGATGATGAAATAATTGCTAAAAGAACTGCTGTGCTCTGCAGGATTGCGGAGGAGTGTTTTGAGAAGATTTCTATAAACAATCCCTATATTACAAGGCCAGTTTATGTGATAGGAGCTGATGTGCCACCTCCCGGCGGAGAGTCTTCTATTTGTCAAACAATTACTACTAAAGATGAATTAGAAAGAAGTTTAGAATATTTCAAAGAAGCATTTAAAAAGGAAGGAATTGAGCATGTATTCGATTATGTAGTTGCTGTTGTTGCAAATTTTGGAGTTGAATTTGGGAGCGATGAAATTGTTGATTTTGATATGGAAAAAGTAAAGCCGCTAAAAGAACTTTTGGCAAAGTACAATATAGTATTTGAAGGCCATTCTACAGATTATCAAACAAAAGAAAACTTAAAAAGAATGGTCGAATGTGGTATTGCAATTTTAAAGGTTGGTCCTGCTCTAACATTTACATTGCGCGAAGCGTTAGTAGCACTTAGTCATATTGAAGAAGAAATTTATAGCAATGAAAAGGAGAAACTGTCAAGATTTAGAGAAGTTTTATTGAATACTATGCTAACATGCAAAGATCACTGGAGTAAATATTTTGATGAGAATGATAAGTTAATTAAGTCAAAGCTCCTATATAGCTATCTTGACAGATGGAGATACTATTTTGAAAACGAGAGTGTGAAAAGTGCTGTTTATTCTCTTATTGGAAATTTAGAGAATGTTAAAATTCCACCTTGGCTTGTAAGTCAGTATTTTCCTTCTCAGTACCAAAAGATGAGAAAAAAAGATTTAAAAAACGGTGCTGCCGACCTAATATTGGATAAAATAGGGGAAGTCATTGACCATTATGTTTATGCGGTAAAAGAAT AA Amino acid sequence(SEQ ID NO.: 4) MSPQNPLIGLFKNREKEFKGIISVCSSNEIVLEAVLKRMKDTNLPIIIEATANQVNQFGGYSGLTPSQFKERVIKIAQKVDFPLERIILGGDHLGPFVWRDQEPEIAMEYAKQMIKEYIKAGFTKIHIDTSMPLKGENSIDDEIIAKRTAVLCRIAEECFEKISINNPYITRPVYVIGADVPPPGGESSICQTITTKDELERSLEYFKEAFKKEGIEHVFDYVVAVVANFGVEFGSDEIVDFDMEKVKPLKELLAKYNIVFEGHSTDYQTKENLKRMVECGIAILKVGPALTFTLREALVALSHIEEEIYSNEKEKLSRFREVLLNTMLTCKDHWSKYFDENDKLIKSKLLYSYLDRWRYYFENESVKSAVYSLIGNLENVKIPPWLVSQYFPSQYQKMRKKDLKNGAADLILD KIGEVIDHYVYAVKE

Thermophilic F6PE from Caldilinea aerophila (Uniprot ID: 101507)

Nucleotide sequence (SEQ ID NO.: 5)ATGTCAACACTTCGCCACATCATTTTGCGACTGATCGAGCTGCGTGAACGAGAACAGATCCATCTCACGCTGCTGGCCGTCTGTCCCAACTCGGCGGCGGTGCTGGAGGCAGCGGTGAAGGTCGCCGCGCGCTGCCACACGCCGATGCTCTTCGCTGCCACGCTCAATCAAGTCGATCGCGACGGCGGCTACACCGGTTGGACGCCTGCGCAATTCGTCGCCGAGATGCGTCGCTATGCCGTCCGCTATGGCTGCACCACCCCGCTCTATCCTTGCCTGGATCACGGCGGGCCGTGGCTCAAAGATCGCCATGCACAGGAAAAGCTACCGCTCGACCAGGCGATGCATGAGGTCAAGCTGAGCCTCACCGCCTGTCTGGAGGCCGGCTACGCGCTGCTGCACATCGACCCCACGGTCGATCGCACGCTCCCGCCCGGAGAAGCGCCGCTCGTGCCGATCGTCGTCGAGCGCACGGTCGAGCTGATCGAACATGCCGAACAGGAGCGACAGCGGCTGAACCTGCCGGCGGTCGCCTATGAAGTCGGCACCGAAGAAGTACATGGCGGGCTGGTGAATTTCGACAATTTTGTCGCCTTCTTGGATTTGCTCAAGGCAAGGCTTGAACAACGTGCCCTGATGCACGCCTGGCCCGCCTTCGTGGTGGCGCAGGTCGGCACTGACCTGCATACAACGTATTTTGACCCCAGTGCGGCGCAACGGCTGACTGAGATCGTGCGCCCTACCGGTGCACTGTTGAAGGGGCACTACACCGACTGGGTCGAAAATCCCGCCGACTATCCGAGGGTAGGCATGGGAGGCGCCAACGTTGGTCCAGAGTTTACGGCGGCCGAGTTCGAGGCGCTGGAAGCGCTGGAACGGCGGGAACAACGGCTGTGCGCCAACCGGAAATTGCAGCCCGCCTGTTTTTTGGCTGCACTGGAAGAGGCAGTAGTCGCTTCAGATCGTTGGCGGAAGTGGCTCCAGCCCGATGAGATCGGCAAGCCCTTTGCAGAATTAACGCCCGCACGCCGGCGCTGGCTCGTGCAGACCGGGGCACGCTACGTCTGGACTGCGCCGAAAGTTATCGCCGCACGCGAACAGCTCTATGCGCACCTCTCCCTTGTGCAGGCGGATCCACATGCCTACGTGGTAGAGTCAGTCGCCCGGTCAATCGAGCGCTATATCGATGCCTTCAACTTATACGACGCCGCTACATTGCTTGGATGA Amino acid sequence (SEQ ID NO.: 6)MSTLRHIILRLIELREREQIHLTLLAVCPNSAAVLEAAVKVAARCHTPMLFAATLNQVDRDGGYTGWTPAQFVAEMRRYAVRYGCTTPLYPCLDHGGPWLKDRHAQEKLPLDQAMHEVKLSLTACLEAGYALLHIDPTVDRTLPPGEAPLVPIVVERTVELIEHAEQERQRLNLPAVAYEVGTEEVHGGLVNFDNFVAFLDLLKARLEQRALMHAWPAFVVAQVGTDLHTTYFDPSAAQRLTEIVRPTGALLKGHYTDWVENPADYPRVGMGGANVGPEFTAAEFEALEALERREQRLCANRKLQPACFLAALEEAVVASDRWRKWLQPDEIGKPFAELTPARRRWLVQTGARYVWTAPKVIAAREQLYAHLSLVQADPHAYVVESVARSIERYIDAFNLYDAATLLG

Thermophilic F6PE from Caldithrix abyssi (Uniprot ID: H1XRG1)

Nucleotide sequence (SEQ ID NO.: 7)ATGAGTCTGCATCCTTTAAATAAATTAATCGAGCGACACAAAAAAGGAACGCCGGTCGGTATTTATTCCGTCTGTTCGGCCAATCCCTTTGTTTTGAAAGCGGCCATGCTACAGGCGCAAAAGGATCAGTCTTTGCTACTTATTGAGGCCACTTCCAACCAGGTAGATCAATTCGGCGGTTACACCGGCATGCGGCCCGAAGATTTTAAAACAATGACGCTTGAACTGGCAGCCGAAAACAATTACGATCCACAGGGATTAATCCTGGGCGGCGACCATCTGGGGCCCAACCGCTGGACAAAACTGAGCGCCTCCCGGGCCATGGACTACGCCAGAGAGCAGATTGCCGCTTATGTTAAAGCCGGCTTTTCCAAAATCCACTTAGACGCCACCATGCCCTTGCAAAACGATGCCACAGATTCCGCCGGCCGCCTTCCAGTCGAAACAATCGCTCAACGTACCGCAGAATTATGCGCCGTGGCCGAACAAACTTACCGGCAGAGCGACCAACTCTTTCCGCCGCCTGTTTACATTGTCGGCAGCGACGTGCCCATCCCGGGCGGCGCGCAAGAAGCGCTGAACCAGATCCATATTACGGAGGTAAAAGAGGTTCAACAGACCATTGATCACGTGCGGCGGGCCTTTGAAAAAAACGGCCTGGAAGCGGCTTACGAAAGAGTTTGCGCCGTTGTCGTGCAGCCAGGCGTTGAATTCGCCGATCAAATCGTTTTTGAATACGCTCCCGACAGAGCGGCGGCCTTAAAAGATTTTATTGAAAGCCATTCGCAGCTGGTTTATGAAGCGCACTCTACTGATTACCAGACCGCACCTCTTTTGCGCCAGATGGTAAAAGATCACTTTGCCATTTTAAAGGTCGGGCCTGCGCTCACCTTTGCCCTGCGCGAAGCCATTTTTGCTCTGGCCTTTATGGAAAAAGAGCTTTTGCCATTGCACAGAGCGCTCAAACCTTCTGCCATTCTGGAAACGCTGGACCAAACGATGGACAAAAACCCTGCTTACTGGCAAAAGCATTACGGCGGAACAAAGGAAGAAGTACGCTTTGCGCAGCGGTTTAGCCTGAGCGACCGCATTCGTTACTACTGGCCGTTTCCAAAGGTTCAAAAGGCCCTGCGCCAATTGCTAAAAAACTTGCAACAAATTTCCATTCCTCTAACTTTGGTAAGCCAGTTCATGCCAGAGGAATACCAACGTATTCGCCAAGGAACGTTAACCAACGATCCGCAGGCGCTGATTTTGAACAAAATTCAAAGCGTATTAAAGCAATACGCGGAGGCGACGCAAATTCAAAACTCTTTGACATTCACGCAAAATCAAAATTCATTAGCAATGGAGCGACTATGA Amino acid sequence (SEQ ID NO.: 8)MSLHPLNKLIERHKKGTPVGIYSVCSANPFVLKAAMLQAQKDQSLLLIEATSNQVDQFGGYTGMRPEDFKTMTLELAAENNYDPQGLILGGDHLGPNRWTKLSASRAMDYAREQIAAYVKAGFSKIHLDATMPLQNDATDSAGRLPVETIAQRTAELCAVAEQTYRQSDQLFPPPVYIVGSDVPIPGGAQEALNQIHITEVKEVQQTIDHVRRAFEKNGLEAAYERVCAVVVQPGVEFADQIVFEYAPDRAAALKDFIESHSQLVYEAHSTDYQTAPLLRQMVKDHFAILKVGPALTFALREAIFALAFMEKELLPLHRALKPSAILETLDQTMDKNPAYWQKHYGGTKEEVRFAQRFSLSDRIRYYWPFPKVQKALRQLLKNLQQISIPLTLVSQFMPEEYQRIRQGTLTNDPQALILNKIQSVLKQYAEATQIQNSLTFTQNQNSLAMERL

Thermophilic F6PE from Dictyoglomus thermophilum (Uniprot ID: B5YBD7)

Nucleotide sequence (SEQ ID NO.: 9)ATGTGGCTTAGTAAAGATTATTTGAGAAAAAAGGGAGTTTATTCTATATGTAGCTCTAATCCATATGTGATTGAGGCAAGTGTTGAATTTGCTAAGGAGAAGAATGATTATATTTTAATTGAGGCGACACCTCATCAGATAAACCAGTTTGGTGGATATTCAGGTATGACTCCCGAAGATTTTAAAAACTTTGTAATGGGAATAATAAAAGAAAAGGGAATAGAAGAGGATAGGGTGATTCTTGGAGGGGACCATTTAGGCCCTCTCCCTTGGCAAGATGAACCTTCTTCTTCTGCAATGAAAAAGGCAAAAGACCTTATAAGGGCCTTTGTGGAGAGTGGTTATAAGAAGATACACCTTGATTGTAGTATGTCTCTTTCTGATGATCCTGTAGTGCTCTCTCCCGAGAAGATAGCAGAAAGGGAGAGGGAACTTCTTGAGGTTGCAGAAGAGACTGCTAGAAAGTACAATTTTCAGCCTGTGTATGTGGTGGGAACTGATGTACCGGTAGCTGGAGGAGGCGAAGAGGAAGGTATTACCTCAGTGGAGGATTTTAGAGTAGCAATCTCCTCTTTAAAAAAATATTTTGAGGATGTTCCAAGGATATGGGATAGGATAATTGGTTTTGTAATAATGCTTGGTATAGGTTTTAATTATGAAAAAGTGTTTGAGTATGACAGGATTAAGGTGAGAAAAATTTTAGAGGAGGTAAAGAAAGAGAATCTTTTTGTTGAAGGTCACTCTACTGACTATCAGACAAAACGTGCATTGAGAGATATGGTAGAGGATGGAGTAAGAATTCTTAAGGTTGGTCCTGCTTTAACAGCAAGTTTTAGAAGGGGAGTATTTTTATTAAGTAGCATTGAGGATGAGCTTATATCGGAAGATAAAAGGTCTAATATTAAGAAAGTTGTGCTTGAGACTATGTTAAAAGATGATAAATATTGGAGAAAGTATTATAAGGATTCAGAAAGATTAGAATTAGATATTTGGTACAACTTACTTGATAGGATTAGATATTATTGGGAATATAAAGAGATAAAAATAGCTTTAAATAGGCTTTTTGAAAATTTTTCGGAAGGGGTTGATATTAGATACATCTATCAATATTTTTATGATTCGTATTTTAAAGTAAGAGAAGGAAAAATAAGAAATGATCCAAGGGAGCTAATAAAGAATGAAATAAAGAAGGTCTTGGAGGACTATCACTATGCTGTAAACTTATAA Codon optimized nucleotide sequence(SEQ ID NO: 10) ATGTGGCTGAGCAAGGACTACCTGCGTAAGAAGGGCGTTTACAGCATTTGCAGCAGCAACCCGTATGTTATTGAAGCGAGCGTGGAGTTCGCGAAGGAGAAAAACGATTACATCCTGATTGAAGCGACCCCGCACCAGATCAACCAATTTGGTGGCTATAGCGGCATGACCCCGGAGGACTTCAAGAACTTTGTTATGGGCATCATTAAGGAAAAAGGTATCGAGGAAGACCGTGTGATTCTGGGTGGCGATCACCTGGGTCCGCTGCCGTGGCAGGATGAGCCGAGCAGCAGCGCGATGAAGAAAGCGAAAGACCTGATCCGTGCGTTCGTTGAAAGCGGTTACAAGAAAATTCACCTGGATTGCAGCATGAGCCTGAGCGACGATCCGGTGGTTCTGAGCCCGGAGAAGATCGCGGAACGTGAGCGTGAACTGCTGGAAGTTGCGGAGGAAACCGCGCGTAAATACAACTTTCAACCGGTGTATGTGGTGGGTACCGATGTTCCGGTTGCGGGTGGCGGTGAGGAAGAGGGTATCACCAGCGTGGAGGACTTCCGTGTTGCGATTAGCAGCCTGAAGAAATACTTTGAAGACGTTCCGCGTATTTGGGATCGTATCATTGGTTTCGTGATCATGCTGGGCATTGGTTTCAACTACGAGAAGGTGTTTGAATATGATCGTATCAAAGTGCGTAAAATTCTGGAAGAGGTTAAGAAAGAGAACCTGTTTGTGGAAGGCCACAGCACCGACTATCAGACCAAGCGTGCGCTGCGTGACATGGTGGAGGATGGCGTTCGTATCCTGAAAGTGGGTCCGGCGCTGACCGCGAGCTTCCGTCGTGGTGTGTTTCTGCTGAGCAGCATCGAGGACGAACTGATTAGCGAGGATAAACGTAGCAACATTAAGAAAGTGGTTCTGGAAACCATGCTGAAGGACGATAAATACTGGCGTAAGTACTATAAAGACAGCGAGCGTCTGGAACTGGATATCTGGTACAACCTGCTGGACCGTATTCGTTACTACTGGGAGTACAAGGAAATCAAGATTGCGCTGAACCGTCTGTTCGAGAACTTTAGCGAAGGCGTTGATATCCGTTACATCTACCAATACTTCTACGACAGCTACTTCAAAGTGCGTGAGGGTAAAATCCGTAACGACCCGCGTGAACTGATTAAGAACGAGATTAAGAAAGTGCTGGAAGACTACCATTATGCGGTGAACCTGTAA Amino acid sequence (SEQ ID NO.: 11)MWLSKDYLRKKGVYSICSSNPYVIEASVEFAKEKNDYILIEATPHQINQFGGYSGMTPEDFKNFVMGIIKEKGIEEDRVILGGDHLGPLPWQDEPSSSAMKKAKDLIRAFVESGYKKIHLDCSMSLSDDPVVLSPEKIAERERELLEVAEETARKYNFQPVYVVGTDVPVAGGGEEEGITSVEDFRVAISSLKKYFEDVPRIWDRIIGFVIMLGIGFNYEKVFEYDRIKVRKILEEVKKENLFVEGHSTDYQTKRALRDMVEDGVRILKVGPALTASFRRGVFLLSSIEDELISEDKRSNIKKVVLETMLKDDKYWRKYYKDSERLELDIWYNLLDRIRYYWEYKEIKIALNRLFENFSEGVDIRYIYQYFYDSYFKVREGKIRNDPRELIKNEIKKVLEDYHYAVNL

Tagatose 6-phosphate phosphatase (T6PP) from Archaeoglobus fugidis(Uniprot ID O29805) was used. Activity was measured in 50 mM HEPESbuffer (pH 7.2) containing 5 mM MgCl₂ and 10 mM T6P at 50° C. Thereaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). Tagatose production was determined bydetecting free phosphate release as described for F6PE.

Thermophilic T6PP from Archaeoglobus fulgidus (Uniprot ID:O29805)

Nucleotide sequence (SEQ ID NO.: 12)ATGTTCAAACCAAAGGCCATCGCAGTTGACATAGATGGCACCCTCACCGACAGAAAGAGGGCTCTGAACTGCAGGGCTGTTGAAGCTCTCCGCAAGGTAAAAATTCCCGTGATTTTGGCCACTGGTAACATATCTTGTTTTGCGAGGGCTGCAGCAAAGCTGATTGGAGTCTCAGACGTGGTAATCTGCGAGAATGGGGGCGTGGTGAGGTTCGAGTACGATGGGGAGGATATTGTTTTAGGAGATAAAGAGAAATGCGTTGAGGCTGTGAGGGTGCTTGAGAAACACTATGAGGTTGAGCTGCTGGACTTCGAATACAGGAAGTCGGAAGTGTGCATGAGGAGGAGCTTTGACATCAACGAGGCGAGAAAGCTCATTGAGGGGATGGGGGTTAAGCTTGTGGATTCAGGCTTTGCCTACCACATTATGGATGCTGATGTTAGCAAGGGAAAAGCTTTGAAGTTCGTTGCCGAGAGGCTTGGTATCAGTTCAGCGGAGTTTGCAGTTATCGGCGACTCAGAGAACGACATAGACATGTTCAGAGTTGCTGGATTCGGAATTGCTGTTGCCAATGCCGATGAGAGGCTGAAGGAGTATGCTGATTTAGTTACGCCATCACCAGACGGCGAGGGGGTTGTTGAGGCTTTGCAGTTTCTGGGATTGTTGCGGTGA Amino acid sequence (SEQ ID NO.: 13)MFKPKAIAVDIDGTLTDRKRALNCRAVEALRKVKIPVILATGNISCFARAAAKLIGVSDVVICENGGVVRFEYDGEDIVLGDKEKCVEAVRVLEKHYEVELLDFEYRKSEVCMRRSFDINEARKLIEGMGVKLVDSGFAYHIMDADVSKGKALKFVAERLGISSAEFAVIGDSENDIDMFRVAGFGIAVANADERLKEYADLVTPSPDGEGVVEALQFLGLLR

T6PP from Archaeoglobus profundus (Uniprot ID D2RHV2_ARCPA)

Nucleotide sequence (SEQ ID NO.: 14)GTGTTCAAGGCTTTGGTAGTTGATATAGACGGAACTTTGACGGATAAGAAGAGGGCAATAAACTGCAGAGCGGTCGAAGCACTTAGAAAACTAAAGATTCCTGTTGTCTTGGCAACCGGAAACATTTCATGCTTTGCAAGGGCTGTAGCTAAGATTATAGGTGTTTCCGATATTGTAATAGCTGAGAACGGAGGTGTTGTCAGATTCAGCTACGACGGAGAGGACATAGTTCTGGGGGATAGAAGTAAATGCTTAAGAGCTTTGGAGACACTTAGAAAACGCTTCAAAGTAGAGCTTCTCGACAACGAATATAGGAAGTCTGAGGTCTGCATGAGGAGGAACTTCCCTATAGAGGAAGCTAGAAAGATACTGCCAAAAGATGTTAGAATAGTCGATACAGGCTTCGCATACCACATAATCGATGCAAATGTCAGCAAGGGGAAGGCTTTGATGTTCATAGCCGATAAGCTTGGCTTGGACGTTAAGGATTTCATTGCGATAGGTGATTCCGAAAACGACATTGAAATGTTGGAAGTTGCAGGTTTTGGCGTTGCAGTTGCGAATGCGGATGAAAAGCTTAAGGAGGTAGCGGATTTGGTCACATCGAAGCCTAATGGAGACGGAGTTGTCGAAGCTCTTGAGTT CTTGGGACTCATTTAGAmino acid sequence (SEQ ID NO.: 15)MFKALVVDIDGTLTDKKRAINCRAVEALRKLKIPVVLATGNISCFARAVAKIIGVSDIVIAENGGVVRFSYDGEDIVLGDRSKCLRALETLRKRFKVELLDNEYRKSEVCMRRNFPIEEARKILPKDVRIVDTGFAYHIIDANVSKGKALMFIADKLGLDVKDFIAIGDSENDIEMLEVAGFGVAVANADEKLKEVADLVTSKPNGDGVVEALEFLGLI

T6PP from Archaeoglobus veneficus (Uniprot ID F2KMK2_ARCVS)

Nucleotide sequence (SEQ ID NO.: 16)ATGCTCCGTCCAAAGGGTCTCGCCATTGACATCGACGGAACCATAACATACAGGAATCGAAGCCTGAACTGTAAGGCCGTTGAAGCTCTCAGGAAGGTAAAAATCCCTGTAGTTCTTGCAACTGGCAACATATCCTGTTTCGCAAGAACTGCTGCAAAGCTTATAGGCGTCTCAGACATTGTTATATGCGAAAATGGAGGTATTGTTCGATTCAGCTACGATGGCGACGACATAGTGCTTGGGGACATAAGCAAATGCCTTAAAGCGGCTGAAATTCTCAAAGAGTACTTTGAAATCGAATTCCTTGACGCTGAGTACAGGAAGTCGGAGGTCTGTCTTCGCAGAAACTTTCCTATTGAAGAGGCGAGGAAAATTCTTCACGATGCAAAGCTTGATGTTAAAATCGTCGATTCAGGTTTTGCGTACCACATAATGGATGCGAAGGTCAGCAAAGGAAGGGCTCTTGAGTACATAGCTGATGAACTTGGTATAAGTCCGAAGGAGTTCGCTGCAATTGGTGATTCTGAGAACGACATAGACCTGATTAAGGCTGCCGGCCTCGGTATTGCCGTTGGAGATGCTGACTTAAAGCTCAAAATGGAGGCCGACGTGGTAGTCTCGAAGAAGAATGGCGATGGAGTTGTTGAAGCACTTGAGCTTCTGGGCTTAATTTAA Amino acid sequence (SEQ ID NO.: 17)MLRPKGLAIDIDGTITYRNRSLNCKAVEALRKVKIPVVLATGNISCFARTAAKLIGVSDIVICENGGIVRFSYDGDDIVLGDISKCLKAAEILKEYFEIEFLDAEYRKSEVCLRRNFPIEEARKILHDAKLDVKIVDSGFAYHIMDAKVSKGRALEYIADELGISPKEFAAIGDSENDIDLIKAAGLGIAVGDADLKLKMEADVVVSKKNGDGVVEALELLGLI

The recombinant cellodextrin phosphorylase and cellobiose phosphorylasefrom C. thermocellum are described in Ye et al. Spontaneous high-yieldproduction of hydrogen from cellulosic materials and water catalyzed byenzyme cocktails. ChemSusChem 2009; 2:149-152. Their activities wereassayed as described.

The recombinant polyphosphate glucokinase from Thermobifida fusca YX isdescribed in Liao et al., One-step purification and immobilization ofthermophilic polyphosphate glucokinase from Thermobifida fusca YX:glucose-6-phosphate generation without ATP. Appl. Microbiol. Biotechnol.2012; 93:1109-1117. Its activities were assayed as described.

The recombinant isoamylase from Sulfolobus tokodaii is described inCheng et al., Doubling power output of starch biobattery treated by themost thermostable isoamylase from an archaeon Sulfolobus tokodaii.Scientific Reports 2015; 5:13184. Its activities were assayed asdescribed.

The recombinant 4-alpha-glucanoltransferase from Thermococcus litoralisis described in Jeon et al. 4-α-Glucanotransferase from theHyperthermophilic Archaeon Thermococcus Litoralis. Eur. J. Biochem.1997; 248:171-178. Its activity was measured as described.

Sucrose phosphorylase from Caldithrix abyssi (Uniprot H1XT50) was used.Its activity was measured in 50 mM HEPES buffer (pH 7.5) containing 10mM sucrose and 12 mM organic phosphate. Glucose 1-phosphate (G1P) wasmeasured using a glucose hexokinase/G6PDH assay kit supplemented with 25U/mL phosphoglucomutase as with alpha-glucan phosphorylase.

Enzyme units used in each Example below can be increased or decreased toadjust the reaction time as desired. For example, if one wanted toperform Example 9 in 8 h instead of 24 h, the units of the enzymes wouldbe increased about 3-fold. Conversely, if one wanted perform example 9in 48 h instead of 24 h the enzyme units could be decreased about2-fold. These examples illustrate how the amount of enzyme units can beused to increase or decrease reaction time while maintaining constantproductivity.

Example 1

To validate the technical feasibility of the enzymatic biosynthesis offructose 6-phosphate from starch, three enzymes were recombinantlyexpressed: alpha-glucan phosphorylase from T. maritima (Uniprot IDG4FEH8), phosphoglucomutase from Thermococcus kodakaraensis (Uniprot IDQ68BJ6), and phosphoisomerase from Clostridium thermocellum (Uniprot IDA3DBX9). The recombinant proteins were over-expressed in E. coli BL21(DE3) and purified as described above.

A 0.20 mL reaction mixture containing 10 g/L soluble starch, 50 mMphosphate buffered saline pH 7.2, 5 mM MgCl₂, 0.5 mM ZnCl₂, 0.01 U ofαGP, 0.01 U PGM, and 0.01 U PGI was incubated at 50° C. for 24 hours.The reaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). The product, fructose 6-phosphate (F6P), wasdetermined using a fructose 6-phosphate kinase (F6PK)/pyruvatedehydrogenase (PK)/lactate dehydrogenase (LD) coupled enzyme assay wherea decrease in absorbance at 340 nm indicates production of F6P asdescribed above. The final concentration of F6P after 24 hours was 3.6g/L.

Example 2

Same tests as in Example 1 (other than reaction temperatures) werecarried out from 40 to 80° C. It was found that 10 g/L soluble starchproduced 0.9 g/L F6P at 40° C. and 3.6 g/L F6P at 80° C. after 40 hourreactions. These results suggest that increasing reaction temperaturefor this set of enzymes increased F6P yields, but too high temperaturemay impair some enzyme activity.

Example 3

It was found that, at 80° C., an enzyme ratio of αGP:PGM:PGI ofapproximately 1:1:1 resulted in fast F6P generation. It was noted thatthe enzyme ratio did not influence final F6P concentration greatly ifthe reaction time was long enough. However, the enzyme ratio affectsreaction rates and the total cost of enzymes used in the system.

Example 4

A 0.20 mL reaction mixture containing 10 g/L maltodextrin, 50 mMphosphate buffered saline pH 7.2, 5 mM MgCl₂, 0.5 mM ZnCl₂, 0.01 U ofαGP, 0.01 U PGM, and 0.01 U PGI was incubated at 50° C. for 24 hours.The reaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). The product, fructose 6-phosphate (F6P), wasdetermined using a fructose 6-phosphate kinase (F6PK)/pyruvatedehydrogenase (PK)/lactate dehydrogenase (LD) coupled enzyme assay wherea decrease in absorbance at 340 nm indicates production of F6P asdescribed above. The final concentration of F6P after 24 hours was 3.6g/L.

Example 5

To test for F6P production from Avicel, Sigma cellulase was used tohydrolyze cellulose at 50° C. To remove beta-glucosidase from commercialcellulase, 10 filter paper units/mL of cellulase was mixed to 10 g/LAvicel at an ice-water bath for 10 min. After centrifugation at 4° C.,the supernatant containing beta-glucosidase was decanted. Avicel thatwas bound with cellulase containing endoglucanase and cellobiohydrolasewas resuspended in a citrate buffer (pH 4.8) for hydrolysis at 50° C.for three days. The cellulose hydrolysate was mixed with 5 U/mLcellodextrin phosphorylase, 5 U/L cellobiose phosphorylase, 5 U/mL ofαGP, 5 U/mL PGM, and 5 U/mL PGI in a 100 mM HEPES buffer (pH 7.2)containing 10 mM phosphate, 5 mM MgCl₂ and 0.5 mM ZnCl₂. The reactionwas conducted at 60° C. for 72 hours and high concentrations of F6P werefound (small amounts of glucose and no cellobiose). F6P was detectedusing the coupled enzyme assay described above. Glucose was detectedusing a hexokinase/G6PDH assay kit as described above.

Example 6

To increase F6P yields from Avicel, Avicel was pretreated withconcentrated phosphoric acid to produce amorphous cellulose (RAC), asdescribed in Zhang et al. A transition from cellulose swelling tocellulose dissolution by o-phosphoric acid: evidence from enzymatichydrolysis and supramolecular structure. Biomacromolecules 2006;7:644-648. To remove beta-glucosidase from commercial cellulase, 10filter paper units/mL of cellulase was mixed with 10 g/L RAC in anice-water bath for 5 min. After centrifugation at 4° C., the supernatantcontaining beta-glucosidase was decanted. The RAC that was bound withcellulase containing endoglucanase and cellobiohydrolase was resuspendedin a citrate buffer (pH 4.8) for hydrolysis at 50° C. for 12 hours. TheRAC hydrolysate was mixed with 5 U/mL cellodextrin phosphorylase, 5 U/Lcellobiose phosphorylase, 5 U/mL of αGP, 5 U/mL PGM, and 5 U/mL PGI in a100 mM HEPES buffer (pH 7.2) containing 10 mM phosphate, 5 mM MgCl₂ and0.5 mM ZnCl₂. The reaction was conducted at 60° C. for 72 hours. Highconcentrations of F6P and glucose were recovered because no enzymes wereadded to convert glucose to F6P. F6P was detected using the coupledenzyme assay described above. Glucose was detected using ahexokinase/G6PDH assay kit as described above.

Example 7

To further increase F6P yields from RAC, polyphosphate glucokinase andpolyphosphate were added. To remove beta-glucosidase from commercialcellulase, 10 filter paper units/mL of cellulase was mixed with 10 g/LRAC in an ice-water bath for 5 min. After centrifugation at 4° C., thesupernatant containing beta-glucosidase was decanted. The RAC that wasbound with cellulase containing endoglucanase and cellobiohydrolase wasre-suspended in a citrate buffer (pH 4.8) for hydrolysis at 50° C. wasincubated in a citrate buffer (pH 4.8) for hydrolysis at 50° C. for 12hours. The RAC hydrolysate was mixed with 5 U/mL polyphosphateglucokinase, 5 U/mL cellodextrin phosphorylase, 5 U/mL cellobiosephosphorylase, 5 U/mL of αGP, 5 U/mL PGM, and 5 U/mL PGI in a 100 mMHEPES buffer (pH 7.2) containing 50 mM polyphosphate, 10 mM phosphate, 5mM MgCl₂ and 0.5 mM ZnCl₂. The reaction was conducted at 50° C. for 72hours. F6P was found in high concentrations with only small amounts ofglucose now present. F6P was detected using the coupled enzyme assaydescribed above. Glucose was detected using a hexokinase/G6PDH assay kitas described above.

Example 8

To validate tagatose production from F6P, 2 g/L F6P was mixed with 1U/ml fructose 6-phosphate epimerase (F6PE) and 1 U/ml tagatose6-phosphate phosphatase (T6PP) in 50 mM HEPES buffer (pH 7.2) containing5 mM MgCl₂. The reaction was incubated for 16 hours at 50° C. 100%conversion of F6P to tagatose is seen via HPLC (Agilent 1100 series)using an Agilent Hi-Plex H-column and refractive index detector. Thesample was run in 5 mM H₂SO₄ at 0.6 mL/min.

Example 9

To validate production of tagatose from maltodextrin, a 0.20 mL reactionmixture containing 20 g/L maltodextrin, 50 mM phosphate buffered salinepH 7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE,and 0.05 U T6PP was incubated at 50° C. for 24 hours. The reaction wasstopped via filtration of enzyme with a Vivaspin 2 concentrator (10,000MWCO). Tagatose was detected and quantified using an Agilent 1100 seriesHPLC with refractive index detector and an Agilent Hi-Plex H-column. Themobile phase was 5 mM H₂SO₄, which ran at 0.6 mL/min. A yield of 9.2 g/Ltagatose was obtained. This equates to 92% of the theoretical yield dueto limits of maltodextrin degradation without enzymes such as isoamylaseor 4-glucan transferase. Standards of various concentrations of tagatosewere used to quantify our yield.

Example 10

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 5.5), 5 mM MgCl₂, and 0.1 g/L isoamylase was incubated at 80° C. for24 hours. This was used to create another reaction mixture containing 20g/L isoamylase treated maltodextrin, 50 mM phosphate buffered saline pH7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, and0.05 U T6PP was incubated at 50° C. for 24 hours. Production of tagatosewas quantified as in Example 9. The yield of tagatose was increased to16 g/L with the pretreatment of maltodextrin by isoamylase. This equatesto 80% of the theoretical yield.

Example 11

To further increase tagatose yields from maltodextrin, 0.05 U 4-glucantransferase (4GT) was added to the reaction described in example 9.

A 0.2 mL reaction mixture containing 20 g/L isoamylase treatedmaltodextrin (see example 9), 50 mM phosphate buffered saline pH 7.2, 5mM MgCl2, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 UT6PP, and 0.05 U 4GT was incubated at 50° C. for 24 hours. Production oftagatose was quantified as in example 9. The yield of tagatose wasincreased to 17.7 g/L with the addition of 4GT to IA-treatedmaltodextrin. This equates to 88.5% of the theoretical yield.

Example 12

To determine the concentration range of phosphate buffered saline (PBS),a 0.20 mL reaction mixture containing 50 g/L maltodextrin; 6.25 mM, 12.5mM, 25 mM, 37.5 mM, or 50 mM phosphate buffered saline pH 7.2; 5 mMMgCl2; 0.1 U of αGP; 0.1 U PGM; 0.1 U PGI; 0.1 U F6PE; and 0.1 U T6PPwas incubated at 50° C. for 6 hours. The short duration ensurescompletion was not reached, and therefore differences in efficiencycould be clearly seen. Production of tagatose was quantified as inexample 9. Respectively, a yield of 4.5 g/L, 5.1 g/L, 5.6 g/L, 4.8 g/L,or 4.9 g/L tagatose was obtained for the reactions containing either6.25 mM, 12.5 mM, 25 mM, 37.5 mM, or 50 mM phosphate buffered saline pH7.2 (Table 1). These results indicate that a concentration of 25 mM PBSpH 7.2 is ideal for these particular reaction conditions. It isimportant to note that even the use of 6.25 mM PBS at pH 7.2 results insignificant turnover due to phosphate recycling. This shows that thedisclosed phosphate recycling methods are able to keep phosphate levelslow even at industrial levels of volumetric productivity (e.g., 200-300g/L maltodextrin).

TABLE 1 Concentration of PBS pH 7.2 (mM) g/L of Tagatose 6.25 4.5 12.55.1 25 5.6 37.5 4.8 50 4.9

Example 13

To determine the pH range of the cascade reaction, a 0.20 mL reactionmixture containing 50 g/L maltodextrin; 50 mM phosphate buffered salinepH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0 7.2, or 7.3; 5 mM MgCl2; 0.02 U of αGP;0.02 U PGM; 0.02 U PGI; 0.02 U F6PE; and 0.02 U T6PP was incubated at50° C. for 16 hours. The units were lowered to ensure completion was notreached, and therefore differences in efficiency could be clearly seen.Production of tagatose was quantified as in example 8. Respectively, ayield of 4.0 g/L, 4.1 g/L 4.2 g/L, 4.1 g/L, 4.4 g/L, 4.1 g/L, 3.8 g/L or4.0 g/L tagatose was obtained for reactions containing 50 mM phosphatebuffered saline at pH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, or 7.3 (Table2). These results indicate that a pH of 6.8 is ideal for theseparticular reaction conditions, although the system works through a widepH range.

TABLE 2 pH of PBS g/L of Tagatose 6.0 4.0 6.2 4.1 6.4 4.2 6.6 4.1 6.84.4 7.0 4.1 7.2 3.8 7.3 4.0

Example 14

To investigate scale-up, a 20 mL reaction mixture containing 50 g/Lisoamylase treated maltodextrin (see Example 9), 50 mM phosphatebuffered saline pH 7.2, 5 mM MgCl2, 10 U of αGP, 10 U PGM, 10 U PGI, 10U F6PE, and 10 U T6PP was incubated at 50° C. for 24 hours. Productionof tagatose was quantified as in example 8. The yield of tagatose was37.6 g/L at the 20 mL scale and 50 g/L maltodextrin. This equates to 75%of the theoretical yield. These results indicate that scale-up to largerreaction volumes will not result in significant loses of yield.

Example 15

To further increase tagatose yields from maltodextrin, 0.05 U maltosephosphorylase is added to the reaction described in Example 9.

Example 16

To further increase tagatose yields from maltodextrin, 0.05 Upolyphosphate glucokinase and 75 mM polyphosphate is added to thereaction described in Example 9.

Example 17

To produce tagatose from fructose, a reaction mixture containing 10 g/Lfructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2,0.05 U fructose polyphosphate kinase, 0.05 U F6PE, and 0.05 U T6PP isincubated at 50° C. for 24 hours. Production of tagatose is quantifiedas in Example 9.

Example 18

To produce tagatose from glucose, a reaction mixture containing 10 g/Lglucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U F6PE, and 0.05 U T6PPis incubated at 50° C. for 24 hours. Production of tagatose isquantified as in Example 9.

Example 19

To produce tagatose from sucrose, a reaction mixture containing 10 g/Lsucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl2, 0.05 Usucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U F6PE, and 0.05 UT6PP is incubated at 50° C. for 24 hours. Production of tagatose isquantified as in Example 9.

Example 20

To further increase yields of tagatose from sucrose, 75 mM polyphosphateand 0.05 polyphosphate fructokinase is added to the reaction mixture inexample 15. Production of tagatose is quantified as in Example 9.

1. A process for preparing tagatose, the process comprising: convertingfructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) catalyzed by anepimerase; and converting the T6P produced to tagatose catalyzed by aphosphatase.
 2. The process of claim 1, further comprising a step ofconverting glucose 6-phosphate (G6P) to the F6P, wherein the step iscatalyzed by phosphoglucose isomerase (PGI).
 3. The process of claim 2,further comprising the step of converting glucose 1-phosphate (G1P) tothe G6P, wherein the step is catalyzed by phosphoglucomutase (PGM). 4.The process of claim 3, further comprising the step of converting asaccharide to the G1P, wherein the step is catalyzed by at least oneenzyme, wherein the saccharide is selected from the group consisting ofa starch or derivative thereof, cellulose or a derivative thereof andsucrose.
 5. The process of claim 4, wherein at least one enzyme isselected from the group consisting of alpha-glucan phosphorylase (αGP),maltose phosphorylase, sucrose phosphorylase, cellodextrinphosphorylase, cellobiose phosphorylase, and cellulose phosphorylase. 6.The process of claim 4, wherein the saccharide is starch or a derivativethereof selected from the group consisting of amylose, amylopectin,soluble starch, amylodextrin, maltodextrin, maltose, and glucose.
 7. Theprocess of claim 4, further comprising the step of converting starch toa starch derivative wherein the starch derivative is prepared byenzymatic hydrolysis of starch or by acid hydrolysis of starch.
 8. Theprocess of claim 6, wherein 4-glucan transferase (4GT) is added to theprocess.
 9. The process of claim 7, wherein the starch derivative isprepared by enzymatic hydrolysis of starch catalyzed by isoamylase,pullulanase, alpha-amylase, or a combination thereof.
 10. The process ofclaim 1, further comprising: the step of converting fructose to the F6P,wherein the step is catalyzed by at least one enzyme; and optionally,the step of converting sucrose to the fructose, wherein the step iscatalyzed by at least one enzyme.
 11. The process of claim 2, furthercomprising: the step of converting glucose to the G6P, wherein the stepis catalyzed by at least one enzyme, and optionally, the step ofconverting sucrose to the glucose, wherein the step is catalyzed by atleast one enzyme. 12-21. (canceled)